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基本情報
登録情報 | データベース: PDB / ID: 6ye4 | ||||||
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タイトル | Structure of ExbB pentamer from Serratia marcescens by single particle cryo electron microscopy | ||||||
![]() | Biopolymer transport protein ExbB | ||||||
![]() | METAL TRANSPORT / membrane protein / iron uptake / proton transfer / TonB complex | ||||||
機能・相同性 | TonB-system energizer ExbB type-1 / MotA/TolQ/ExbB proton channel / MotA/TolQ/ExbB proton channel family / transmembrane transporter activity / protein transport / membrane => GO:0016020 / plasma membrane / Chem-PGT / Biopolymer transport protein ExbB![]() | ||||||
生物種 | ![]() | ||||||
手法 | 電子顕微鏡法 / 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 3.2 Å | ||||||
![]() | Biou, V. / Delepelaire, P. / Coureux, P.D. / Chami, M. | ||||||
資金援助 | ![]()
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![]() | ![]() タイトル: Structural and molecular determinants for the interaction of ExbB from Serratia marcescens and HasB, a TonB paralog. 著者: Valérie Biou / Ricardo Jorge Diogo Adaixo / Mohamed Chami / Pierre-Damien Coureux / Benoist Laurent / Véronique Yvette Ntsogo Enguéné / Gisele Cardoso de Amorim / Nadia Izadi-Pruneyre / ...著者: Valérie Biou / Ricardo Jorge Diogo Adaixo / Mohamed Chami / Pierre-Damien Coureux / Benoist Laurent / Véronique Yvette Ntsogo Enguéné / Gisele Cardoso de Amorim / Nadia Izadi-Pruneyre / Christian Malosse / Julia Chamot-Rooke / Henning Stahlberg / Philippe Delepelaire / ![]() ![]() ![]() 要旨: ExbB and ExbD are cytoplasmic membrane proteins that associate with TonB to convey the energy of the proton-motive force to outer membrane receptors in Gram-negative bacteria for iron uptake. The ...ExbB and ExbD are cytoplasmic membrane proteins that associate with TonB to convey the energy of the proton-motive force to outer membrane receptors in Gram-negative bacteria for iron uptake. The opportunistic pathogen Serratia marcescens (Sm) possesses both TonB and a heme-specific TonB paralog, HasB. ExbB has a long periplasmic extension absent in other bacteria such as E. coli (Ec). Long ExbB's are found in several genera of Alphaproteobacteria, most often in correlation with a hasB gene. We investigated specificity determinants of ExbB and HasB. We determined the cryo-EM structures of ExbB and of the ExbB-ExbD complex from S. marcescens. ExbB alone is a stable pentamer, and its complex includes two ExbD monomers. We showed that ExbB extension interacts with HasB and is involved in heme acquisition and we identified key residues in the membrane domain of ExbB and ExbB, essential for function and likely involved in the interaction with TonB/HasB. Our results shed light on the class of inner membrane energy machinery formed by ExbB, ExbD and HasB. #1: ![]() タイトル: Functional and structural characterization of Serratia marcescens ExbB: determinants of the interaction with HasB/TonB 著者: Biou, V. / Chami, M. / Coureux, P.D. / Laurent, B. / Ntsogo, Y. / Izadi-Pruneyre, N. / Malosse, C. / Chamot-Rooke, J. / Stahlberg, H. / Delepelaire, P. #2: ジャーナル: J Struct Biol / 年: 2012 タイトル: RELION: implementation of a Bayesian approach to cryo-EM structure determination. 著者: Sjors H W Scheres / ![]() 要旨: RELION, for REgularized LIkelihood OptimizatioN, is an open-source computer program for the refinement of macromolecular structures by single-particle analysis of electron cryo-microscopy (cryo-EM) ...RELION, for REgularized LIkelihood OptimizatioN, is an open-source computer program for the refinement of macromolecular structures by single-particle analysis of electron cryo-microscopy (cryo-EM) data. Whereas alternative approaches often rely on user expertise for the tuning of parameters, RELION uses a Bayesian approach to infer parameters of a statistical model from the data. This paper describes developments that reduce the computational costs of the underlying maximum a posteriori (MAP) algorithm, as well as statistical considerations that yield new insights into the accuracy with which the relative orientations of individual particles may be determined. A so-called gold-standard Fourier shell correlation (FSC) procedure to prevent overfitting is also described. The resulting implementation yields high-quality reconstructions and reliable resolution estimates with minimal user intervention and at acceptable computational costs. | ||||||
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構造の表示
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構造ビューア | 分子: ![]() ![]() |
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-検証レポート
文書・要旨 | ![]() | 1.3 MB | 表示 | ![]() |
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-関連構造データ
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リンク
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集合体
登録構造単位 | ![]()
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要素
#1: タンパク質 | 分子量: 30413.621 Da / 分子数: 5 / 由来タイプ: 組換発現 / 詳細: a 6-histidine tag is present at the C-terminus / 由来: (組換発現) ![]() ![]() ![]() #2: 化合物 | ChemComp-PGT / ( 研究の焦点であるリガンドがあるか | Y | |
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-実験情報
-実験
実験 | 手法: 電子顕微鏡法 |
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EM実験 | 試料の集合状態: PARTICLE / 3次元再構成法: 単粒子再構成法 |
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試料調製
構成要素 | 名称: Homopentamer of ExbB / タイプ: COMPLEX 詳細: the expressed sequence corresponds to the mature sequence after signal peptide cleavage. Entity ID: #1 / 由来: RECOMBINANT | ||||||||||||||||||||
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分子量 | 値: 0.174 MDa / 実験値: NO | ||||||||||||||||||||
由来(天然) | 生物種: ![]() | ||||||||||||||||||||
由来(組換発現) | 生物種: ![]() ![]() | ||||||||||||||||||||
緩衝液 | pH: 8 / 詳細: 20mM Tris-HCl pH 8,0 100mM NaCl 0,0015% LMNG | ||||||||||||||||||||
緩衝液成分 |
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試料 | 濃度: 0.1 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES 詳細: the sample was monodisperse as evidenced by gel filtration column | ||||||||||||||||||||
試料支持 | グリッドの材料: COPPER / グリッドのサイズ: 300 divisions/in. / グリッドのタイプ: Quantifoil R2/4 | ||||||||||||||||||||
急速凍結 | 装置: LEICA EM CPC / 凍結剤: ETHANE / 湿度: 95 % / 凍結前の試料温度: 277 K |
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電子顕微鏡撮影
実験機器 | ![]() モデル: Titan Krios / 画像提供: FEI Company |
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顕微鏡 | モデル: FEI TITAN KRIOS |
電子銃 | 電子線源: ![]() |
電子レンズ | モード: BRIGHT FIELD / 倍率(公称値): 165000 X / 最大 デフォーカス(公称値): 2500 nm / 最小 デフォーカス(公称値): 1500 nm / Cs: 2.7 mm / C2レンズ絞り径: 50 µm |
試料ホルダ | 凍結剤: NITROGEN 試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER 最高温度: 93 K / 最低温度: 83 K |
撮影 | 平均露光時間: 7 sec. / 電子線照射量: 55.95 e/Å2 / 検出モード: SUPER-RESOLUTION フィルム・検出器のモデル: GATAN K2 SUMMIT (4k x 4k) 撮影したグリッド数: 1 / 実像数: 3122 詳細: frames were weighted according to electron dose and particle movement during Relion bayesian polishing procedure. |
電子光学装置 | エネルギーフィルター名称: GIF Quantum LS / エネルギーフィルタースリット幅: 20 eV |
画像スキャン | 横: 3710 / 縦: 3838 / 動画フレーム数/画像: 56 / 利用したフレーム数/画像: 1-56 |
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解析
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画像処理 | 詳細: images were processed with Motioncor2 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
CTF補正 | タイプ: NONE | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
粒子像の選択 | 選択した粒子像数: 1291382 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
対称性 | 点対称性: C5 (5回回転対称) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
3次元再構成 | 解像度: 3.2 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 157111 / アルゴリズム: FOURIER SPACE / クラス平均像の数: 1 / 対称性のタイプ: POINT | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
原子モデル構築 | B value: 55.3 / プロトコル: FLEXIBLE FIT / 空間: REAL / Target criteria: Correlation coefficient 詳細: the ExbB sequence from S. marcescens was modeled by homology from the 5SV0 monomer from E. coli using Phyre software and the pentamer was generated using the 5SV0 symmetry. real space ...詳細: the ExbB sequence from S. marcescens was modeled by homology from the 5SV0 monomer from E. coli using Phyre software and the pentamer was generated using the 5SV0 symmetry. real space refinement was carried out with rigid body, simulated annealing and morphing steps. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
原子モデル構築 | PDB-ID: 5SV0 PDB chain-ID: A / Accession code: 5SV0 / Pdb chain residue range: 10-234 / Source name: PDB / タイプ: experimental model | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
精密化 | 交差検証法: NONE 立体化学のターゲット値: GeoStd + Monomer Library + CDL v1.2 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
原子変位パラメータ | Biso mean: 45.97 Å2 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
拘束条件 |
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